泥鳅Dmrt1基因的克隆、表达和选择性剪接分析
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摘要
果蝇的性别决定基因dsx与线虫的性别决定基因Mab-3的DNA序列比较表明二者具有一个高度同源的区域,称为DM基序。该基序可以与DNA进行序列特异性结合。进一步的研究发现在哺乳类、鸟类、爬行类以及鱼类等脊椎动物中也存在具有DM基序的基因,称为DMRT(Doublesex and Mab-3 related transcription factor,DMRT,Doublesex和Mab-3相关的转录因子)基因家族。其中Dmrt1基因是迄今为止该家族成员中研究最为深入的转录调控因子之一,也是目前发现的第一个在种属间具有进化保守性的性别分化相关基因,在动物性别决定过程中发挥重要作用。鱼类在脊椎动物系统进化中处于承前启后的地位,是性别决定研究的重要载体。因此对鱼类中Dmrt1基因相关信息的研究,不仅有助于了解Dmrt1基因的进化、作用方式及其具体功能等,更有助于揭示鱼类中性别决定及分化体系的运行机制,进而促进阐明脊椎动物的性别决定和分化体系的进化历程。
本研究根据已经公开发表的相关Dmrt1基因资料,针对保守序列设计了一对简并引物,利用RT-PCR的方法克隆出了泥鳅的Dmrt1基因的DM保守盒,并结合RACE技术获得了Dmrt1基因的全长cDNA。结果表明:泥鳅Dmrt1基因外显子的缺失产生了多种拼接形式,共得到六条Dmrt1基因,分别记为MaDmrt1a1(2162 bp)、MaDmrt1a2(2042 bp)、MaDmrt1a3(1993 bp)、MaDmrt1a4(848 bp)、MaDmrt1b(1840 bp)和MaDmrt1c(666 bp),它们都含有DM基序和polyA尾。其中MaDmrt1a1~4具有相同的开放阅读框,包含完整的5个外显子,编码267个氨基酸,其差异主要在于3′非翻译区长度和序列不同;MaDmrt1b缺失第4个外显子,编码219个氨基酸;MaDmrt1c缺失第4和第5个外显子,保留了一段长36bp内含子,编码182个氨基酸。
为了研究两种泥鳅Dmrt1基因在成体不同组织和胚胎发育不同时期的时空表达模式,采用了半定量、实时荧光定量RT-PCR、组织原位杂交和胚胎整体原位杂交技术。结果表明:泥鳅Dmrt1基因只在精巢中强烈表达,而在卵巢中不表达或只有极微弱表达,这与其它物种中的研究结果相一致;在胚胎发育的各个时期泥鳅Dmrt1基因均有表达,但表达强度又各不相同,呈现先上升后下降的表达趋势。综上所述,可推断泥鳅的Dmrt1与雄性性腺分化和发育具有明显的相关性。原位杂交试验结果显示:未成熟的生殖细胞(卵巢的卵原细胞、初级卵母细胞,精巢中的精原细胞和精母细胞)中表达,据此推测泥鳅Dmrt1基因可能调控泥鳅性腺的发育和分化;整体胚胎杂交表明泥鳅Dmrt1基因在体节形成期的体节中大量表达,之后一直到尾芽期的胚胎脊柱中都有很强的阳性杂交信号,孵出期以后Dmrt1基因的表达主要集中在泥鳅的脊柱、体节和头部。这些结果为阐述Dmrt1在脊椎动物体内对性别决定和性别分化的作用奠定了基础。
The DM (Doublesex and Mab-3) domain was identified by the homologous analysis between the sex determining gene doublesex(dsx) in Drosophila melanogaster and Mab-3 in Caenorhabditis.elegans. DM domain-containing genes act as transcription regulators, with the DNA domain conferring sequence-specific zinc finger-like DNA binding. More DM domain-containing genes have been identified from vertebrates including mammals, birds, turtle and fishes. So, a novel family was identified named DMRT (Doublesex and Mab-3 related transcription factor). As one of member in the family, Dmrt1 is an important transcription factor, which research is the most thorough and extensive to date. Dmrt1 is the first conserved gene found among animal phyla for sexual development, which plays an important role during animal sex determination. For fish serves as a link in the phylogenetic evolution of vertebrate, it is an important research carrier of sex determination. So researching it can not only help to reveal the mechanisms of the sex determination and the differentiation system in fish; but also help to expound the evolutional process of the sex determination and the differentiation system in vertebrates.
To isolate the Dmrt1 gene, we amplified the Dmrt1 DM-domain by PCR, using the forward and backward primers designed on the basis of the conserved region amino acid sequence of the Dmrt1 genes registered in Genbank. With the testis cDNA of Misgurnus anguillicaudatus as template, one major fragment(about 140bp) was amplified by RT-PCR. Based on these study, the full-length cDNA of Dmrt1 were cloned from Misgurnus anguillicaudatus using 5'-and 3'-rapid amplification of cDNA ends(RACE). The 5′-and 3′-RACE results were jointed to obtain the full-lengths of DMRT1 cDNA sequences with the DNAMAN software respectively. Six novel isoforms generated by alternative splicing were cloned from Misgurnus anguillicaudatu, which all have the DM domain and 3’polyA tail. In terms of general nomenclature rule, they were named MaDmrt1a1 (2162 bp, encode 267aa), MaDmrt1a2(2042 bp, encode 267aa), MaDmrt1a3(1993 bp, encode 267aa), MaDmrt1a4 (848 bp, encode 267aa), MaDmrt1b (1840 bp, encode 219aa) and MaDmrt1c (666 bp, encode 182aa) respectively. By means of genome comparison, we conjecture that MaDmrt1a have all the five exons, MaDmrt1b lacks exon4, and MaDmrt1c lacks both exon4 and exon5 but uses a 36bp region of intron sequence as its exon 4 at the 3’ends. MaDmrt1a2 , MaDmrt1a3 and MaDmrt1a4 have anterior alternative polyadenylation signals in 3’UTR than Dmrt1a1.
Several technologies, such as Semi-quantitive PCR, ?uorescent quantitative RT-PCR and in situ hybridization, were used to analyse the expression pattern of Dmrt1. The result revealed that the expression of Dmrt1 was testis-specific in adults, with no or very week expression in ovary. This pattern is in line with that of other species’s Dmrt1. Contrary to expectations, all the Dmrt1 transcripts were detectable in all embryos tested. However, take the result by and large, each isoform’s expression trend was risie at first and then decline. Taken together, these results provided the basic data for elucidating Dmrt1 role(s) for sex-determination and gonadal differentiation in vertebrates. The differential expression of these transcripts provides new insight into roles of alternative splicing of Dmrt1 in governing sex differentiation and development of Misgurnus anguillicaudatus. In site hybridization results demonstrated that Dmrt1 express in premature germ cells (ogania, primary oocytes in ovaries, spermatogonias and spermatocytes in testes) of gonads during gonadal development,suggesting roles of Dmrt1 in Misgurnus anguillicaudatu gonads development and differentiation. The zygotic expression of Dmrt1 all occure in the middle of segmentation, and persists throughout tail-bud formed, strong positive hybrid signals could be detected in the spine. Till hatched larva, the major tissues expressing Dmrt1 include notochord, somites and eyes. Taken together, these results provided the basic data for elucidating Dmrt1 role(s) for sex-determination and gonadal differentiation in vertebrates.
本研究根据已经公开发表的相关Dmrt1基因资料,针对保守序列设计了一对简并引物,利用RT-PCR的方法克隆出了泥鳅的Dmrt1基因的DM保守盒,并结合RACE技术获得了Dmrt1基因的全长cDNA。结果表明:泥鳅Dmrt1基因外显子的缺失产生了多种拼接形式,共得到六条Dmrt1基因,分别记为MaDmrt1a1(2162 bp)、MaDmrt1a2(2042 bp)、MaDmrt1a3(1993 bp)、MaDmrt1a4(848 bp)、MaDmrt1b(1840 bp)和MaDmrt1c(666 bp),它们都含有DM基序和polyA尾。其中MaDmrt1a1~4具有相同的开放阅读框,包含完整的5个外显子,编码267个氨基酸,其差异主要在于3′非翻译区长度和序列不同;MaDmrt1b缺失第4个外显子,编码219个氨基酸;MaDmrt1c缺失第4和第5个外显子,保留了一段长36bp内含子,编码182个氨基酸。
为了研究两种泥鳅Dmrt1基因在成体不同组织和胚胎发育不同时期的时空表达模式,采用了半定量、实时荧光定量RT-PCR、组织原位杂交和胚胎整体原位杂交技术。结果表明:泥鳅Dmrt1基因只在精巢中强烈表达,而在卵巢中不表达或只有极微弱表达,这与其它物种中的研究结果相一致;在胚胎发育的各个时期泥鳅Dmrt1基因均有表达,但表达强度又各不相同,呈现先上升后下降的表达趋势。综上所述,可推断泥鳅的Dmrt1与雄性性腺分化和发育具有明显的相关性。原位杂交试验结果显示:未成熟的生殖细胞(卵巢的卵原细胞、初级卵母细胞,精巢中的精原细胞和精母细胞)中表达,据此推测泥鳅Dmrt1基因可能调控泥鳅性腺的发育和分化;整体胚胎杂交表明泥鳅Dmrt1基因在体节形成期的体节中大量表达,之后一直到尾芽期的胚胎脊柱中都有很强的阳性杂交信号,孵出期以后Dmrt1基因的表达主要集中在泥鳅的脊柱、体节和头部。这些结果为阐述Dmrt1在脊椎动物体内对性别决定和性别分化的作用奠定了基础。
The DM (Doublesex and Mab-3) domain was identified by the homologous analysis between the sex determining gene doublesex(dsx) in Drosophila melanogaster and Mab-3 in Caenorhabditis.elegans. DM domain-containing genes act as transcription regulators, with the DNA domain conferring sequence-specific zinc finger-like DNA binding. More DM domain-containing genes have been identified from vertebrates including mammals, birds, turtle and fishes. So, a novel family was identified named DMRT (Doublesex and Mab-3 related transcription factor). As one of member in the family, Dmrt1 is an important transcription factor, which research is the most thorough and extensive to date. Dmrt1 is the first conserved gene found among animal phyla for sexual development, which plays an important role during animal sex determination. For fish serves as a link in the phylogenetic evolution of vertebrate, it is an important research carrier of sex determination. So researching it can not only help to reveal the mechanisms of the sex determination and the differentiation system in fish; but also help to expound the evolutional process of the sex determination and the differentiation system in vertebrates.
To isolate the Dmrt1 gene, we amplified the Dmrt1 DM-domain by PCR, using the forward and backward primers designed on the basis of the conserved region amino acid sequence of the Dmrt1 genes registered in Genbank. With the testis cDNA of Misgurnus anguillicaudatus as template, one major fragment(about 140bp) was amplified by RT-PCR. Based on these study, the full-length cDNA of Dmrt1 were cloned from Misgurnus anguillicaudatus using 5'-and 3'-rapid amplification of cDNA ends(RACE). The 5′-and 3′-RACE results were jointed to obtain the full-lengths of DMRT1 cDNA sequences with the DNAMAN software respectively. Six novel isoforms generated by alternative splicing were cloned from Misgurnus anguillicaudatu, which all have the DM domain and 3’polyA tail. In terms of general nomenclature rule, they were named MaDmrt1a1 (2162 bp, encode 267aa), MaDmrt1a2(2042 bp, encode 267aa), MaDmrt1a3(1993 bp, encode 267aa), MaDmrt1a4 (848 bp, encode 267aa), MaDmrt1b (1840 bp, encode 219aa) and MaDmrt1c (666 bp, encode 182aa) respectively. By means of genome comparison, we conjecture that MaDmrt1a have all the five exons, MaDmrt1b lacks exon4, and MaDmrt1c lacks both exon4 and exon5 but uses a 36bp region of intron sequence as its exon 4 at the 3’ends. MaDmrt1a2 , MaDmrt1a3 and MaDmrt1a4 have anterior alternative polyadenylation signals in 3’UTR than Dmrt1a1.
Several technologies, such as Semi-quantitive PCR, ?uorescent quantitative RT-PCR and in situ hybridization, were used to analyse the expression pattern of Dmrt1. The result revealed that the expression of Dmrt1 was testis-specific in adults, with no or very week expression in ovary. This pattern is in line with that of other species’s Dmrt1. Contrary to expectations, all the Dmrt1 transcripts were detectable in all embryos tested. However, take the result by and large, each isoform’s expression trend was risie at first and then decline. Taken together, these results provided the basic data for elucidating Dmrt1 role(s) for sex-determination and gonadal differentiation in vertebrates. The differential expression of these transcripts provides new insight into roles of alternative splicing of Dmrt1 in governing sex differentiation and development of Misgurnus anguillicaudatus. In site hybridization results demonstrated that Dmrt1 express in premature germ cells (ogania, primary oocytes in ovaries, spermatogonias and spermatocytes in testes) of gonads during gonadal development,suggesting roles of Dmrt1 in Misgurnus anguillicaudatu gonads development and differentiation. The zygotic expression of Dmrt1 all occure in the middle of segmentation, and persists throughout tail-bud formed, strong positive hybrid signals could be detected in the spine. Till hatched larva, the major tissues expressing Dmrt1 include notochord, somites and eyes. Taken together, these results provided the basic data for elucidating Dmrt1 role(s) for sex-determination and gonadal differentiation in vertebrates.
引文
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